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Biomethanation of carbon dioxide (CO 2 ) from flue gas is a potential enabler of the green transition, particularly when integrated with the power-to-gas chain. However, challenges arise in achieving synthetic natural gas quality when utilizing CO 2 from diluted carbon sources, and the high costs of CO 2 separation using amine-based solutions make large-scale implementation unfeasible. We propose an innovative continuous biomethanation system that integrates carbon capture and CO 2 stripping through microbial utilization, eliminating expenses with the stripper. Stable continuous biomethane production (83-92 % methane purity) was achieved from flue gas-CO 2 using a biocompatible aqueous n-methyldiethanolamine (MDEA) solution (50 mmol/L) under mesophilic and hydrogen-limiting conditions. MDEA was found to be recalcitrant to biodegradation and could be reused after regeneration. Demonstrating the microbial ability to simultaneously strip and convert the captured CO 2 and regenerate MDEA provides a new pathway for valorization of flue gas CO 2 ., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michael Vedel Wegener Kofoed reports financial support was provided by Apple Inc. Michael VW Kofoed has patent #PCT/EP2023/069435 pending to Aarhus University. Lars DM Ottosen has patent #PCT/EP2023/069435 pending to Aarhus University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Several epidemiological studies have linked lead (Pb) exposure to induced oxidative stress and the promotion of inflammatory response. We performed a within-subjects study (repeated measures study) to evaluate the relationship between the concentration of blood lead (B-Pb) and toenail lead (T-Pb) and circulating markers of inflammation., Methods: We evaluated the associations between B-Pb concentrations and T-Pb concentrations and circulating markers of inflammation, soluble intracellular adhesion molecule-1 (s-ICAM-1), soluble vascular adhesion molecule-1 (s-VCAM-1), and high-sensitivity C-reactive protein (hs-CRP) on 158 traffic enforcers from the Metropolitan Manila Development Authority (MMDA) traffic enforcer's health study. Linear mixed-effects models with random subject-specific intercepts were fitted to estimate the association between B-Pb and T-Pb exposure and circulating markers of inflammation, adjusting for confounding factors., Results: Traffic enforcers were middle-aged men (89.4%) with a mean age (± SD) of 37.1 years ± 8.9 years and had a total of 293 valid markers of inflammation measurements. B-Pb concentration was related to increased hs-CRP levels. A 10% increase in B-Pb was associated with a 5.7% increase in hs-CRP level [95% confidence interval (95% CI): 1.3-10.1]. However, B-Pb was not associated with s-ICAM-1 and s-VCAM-1. Furthermore, no associations were observed between T-Pb and all the circulating markers of inflammation., Conclusions: Low-level B-Pb may increase hs-CRP among traffic enforcers. Moreover, the study suggests that Pb via the oxidative and inflammation pathways may have an essential role in the development of cardiovascular disease. Furthermore, MMDA and the Department of Labor and Employment can use our study's findings as evidence to conduct routine screening of blood heavy metals, especially Pb, among MMDA and other traffic enforcers as part of their yearly medical examination., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Amphetamine-type stimulants are the third most widely consumed category of illicit drugs worldwide. Faced with the growing problem of amphetamine-type stimulants, numerous qualitative and quantitative techniques have been developed to detect amphetamine (AMP), methamphetamine (MET), MDMA, MDEA or MDA in biological matrices, including hair. Hair analysis is widely used in forensic medicine, but one of its main drawbacks remains external contamination. In this study, we investigated the possibility of hair contamination through external exposure to blood containing AMP, MET MDMA, MDEA or MDA at 2 ng/mL; 20 ng/mL; 200 ng/mL or 2000 ng/mL after 6 h, 1, 3, 7 or 14 days of contact protected from light at room temperature (RT or 20 °C) or at 4 °C. Dried extracts of hair samples were analyzed by UPLC-MS/MS after extensive washings in several baths of water, methanol and acetone before grounding. At the end of our study, contamination of hair was observed from 6 h of contact with all tested amphetamine-type stimulants. The concentrations found in hair ranged from 3 ± 1 to 1464 ± 10 pg/mg, 5 ± 1 to 5070 ± 160 pg/mg, 3 ± 1 to 1269 ± 60 pg/mg, 4 ± 1 to 1860 ± 113 pg/mg and from 8 ± 1 to 1041 ± 44 pg/mg for AMP, MET, MDMA, MDEA and MDA, respectively. Possibly due to its low polar surface area, MET was the most prone to contaminate. As anticipated, hair contamination was mainly dependent on the concentration of all molecules in the contaminating blood, reaching the SOHT cut-off of 200 pg/mg when amphetamine-type stimulants are at toxic or lethal concentrations in the blood. These observations call for caution in interpreting exposure to these substances in such forensic situations., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The rates of CO 2 absorption into fresh and regenerated aqueous solutions of N,N-diethylethanolamine (DEEA), N-methyldiethanolamine (MDEA), and their mixture with sulfolane are investigated in a batch stirred cell reactor. The data are obtained in the temperature range of 293.15-313.15 K, pressures up to 800 kPa, and different concentrations of alkanolamines and sulfolane. The diffusion coefficients and Henry's law constants for all the solutions are obtained. The absorption rate of DEEA solutions increased by increasing component concentrations and pressure, but the effects of temperature on the absorption rates of hybrid and aqueous DEEA solutions are different. Comparison of absorption rates in aqueous and hybrid solutions under the same conditions can determine the role of sulfolane as the physical solvent. It has been found that sulfolane acts as an effective absorption activator in the hybrid DEEA solutions. However, in the MDEA solutions, in all experimental conditions except for high pressure ([Formula: see text] 400 kPa) and certain MDEA concentration (20 wt%), sulfolane has a negative effect on the absorption rate. The absorption rates of regenerated aqueous DEEA solutions are in the range of 50.5-87.7% of fresh ones, while these values for the hybrid DEEA solution are in the range of 75-90.5%. These values for the aqueous and hybrid MDEA solutions are almost equal. Based on the values of Hatta number and enhancement factor, the CO 2 absorption regime in the DEEA solutions is determined as the fast second-order reaction. The absorption rate can be interpreted considering the tradeoff between kinetics and thermodynamics of CO 2 absorption in the aqueous and hybrid DEEA/MDEA solutions. The desorption rates in hybrid DEEA/MDEA solutions are higher than those in aqueous solutions., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Many psychoactive compounds have been shown to primarily interact with high-affinity and low-capacity solute carrier 6 (SLC6) monoamine transporters for norepinephrine (NET; norepinephrine transporter), dopamine (DAT; dopamine transporter) and serotonin (SERT; serotonin transporter). Previous studies indicate an overlap between the inhibitory capacities of substances at SLC6 and SLC22 human organic cation transporters (SLC22A1-3; hOCT1-3) and the human plasma membrane monoamine transporter (SLC29A4; hPMAT), which can be classified as high-capacity, low-affinity monoamine transporters. However, interactions between central nervous system active substances, the OCTs, and the functionally-related PMAT have largely been understudied. Herein, we report data from 17 psychoactive substances interacting with the SLC6 monoamine transporters, concerning their potential to interact with the human OCT isoforms and hPMAT by utilizing radiotracer-based in vitro uptake inhibition assays at stably expressing human embryonic kidney 293 cells (HEK293) cells. Many compounds inhibit substrate uptake by hOCT1 and hOCT2 in the low micromolar range, whereas only a few substances interact with hOCT3 and hPMAT. Interestingly, methylphenidate and ketamine selectively interact with hOCT1 or hOCT2, respectively. Additionally, 3,4-methylenedioxymethamphetamine (MDMA) is a potent inhibitor of hOCT1 and 2 and hPMAT. Enantiospecific differences of R- and S-α-pyrrolidinovalerophenone (R- and S-α-PVP) and R- and S-citalopram and the effects of aromatic substituents are explored. Our results highlight the significance of investigating drug interactions with hOCTs and hPMAT, due to their role in regulating monoamine concentrations and xenobiotic clearance.

Exposure to traffic-related air pollution is linked with acute alterations in blood pressure (BP). We examined the cumulative short-term effect of black carbon (BC) exposure on systolic (SBP) and diastolic (DBP) BP and assessed effect modification by participant characteristics. SBP and DBP were repeatedly measured on 152 traffic enforcers. Using a linear mixed-effects model with random intercepts, quadratic (QCDL) and cubic (CCDL) constrained distributed lag models were fitted to estimate the cumulative effect of BC concentration on SBP and DBP during the 10 hours (daily exposure) and 7 days (weekly exposure) before the BP measurement. Ambient BC was related to increased BP with QCDL models. An interquartile range change in BC cumulative during the 7 days before the BP measurement was associated with increased BP (1.2% change in mean SBP, 95% confidence interval (CI), 0.1 to 2.3; and 0.5% change in mean DBP, 95% CI, -0.8 to 1.7). Moreover, the association between the 10-h cumulative BC exposure and SBP was stronger for female (4.0% change, 95% CI: 2.1-5.9) versus male and for obese (2.9% change, 95% CI: 1.0-4.8) vs. non-obese traffic enforcers. Short-term cumulative exposure to ambient traffic-related BC could bring about cardiovascular diseases through mechanisms involving increased BP.

Synthetic cathinones are among the most popular new psychoactive substances, being abused for their stimulant properties, which are similar to those of amphetamine and 3,4-methylenedioxymethamphetamine (MDMA). Considering that the liver is a likely target for cathinones-induced toxicity, and for their metabolic activation/detoxification, we aimed to determine the hepatotoxicity of three commonly abused synthetic cathinones: butylone, α-methylamino-butyrophenone (buphedrone) and 3,4-dimethylmethcathinone (3,4-DMMC). We characterized their cytotoxic profile in primary rat hepatocytes (PRH) and in the HepaRG and HepG2 cell lines. PRH was the most sensitive cell model, showing the lowest EC 50 values for all three substances (0.158 mM for 3,4-DMMC; 1.21 mM for butylone; 1.57 mM for buphedrone). Co-exposure of PRH to the synthetic cathinones and CYP450 inhibitors (selective and non-selective) proved that hepatic metabolism reduced the toxicity of buphedrone but increased that of butylone and 3,4-DMMC. All compounds were able to increase oxidative stress, disrupting mitochondrial homeostasis and inducing apoptotic and necrotic features, while also increasing the occurrence of acidic vesicular organelles in PRH, compatible with autophagic activation. In conclusion, butylone, buphedrone and 3,4-DMMC have hepatotoxic potential, and their toxicity lies in the interference with a number of homeostatic processes, while being influenced by their metabolic fate.

Phenylalanine ammonia-lyase (PAL) catalyzes the reversible deamination of phenylalanine to cinnamic acid and ammonia. Algae have been considered as biofactories for PAL production, however, biochemical characterization of PAL and its potency for myristicin biotransformation into MMDA (3-methoxy-4, 5-methylenedioxyamphetamine) has not been studied yet. Thus, PAL from Anabaena flos-aquae and Spirulina platensis has been purified, comparatively characterized and its affinity to transform myristicin was assessed. The specific activity of purified PAL from S. platensis (73.9 μmol/mg/min) and A. flos-aquae (30.5 μmol/mg/min) was increased by about 2.9 and 2.4 folds by gel-filtration comparing to their corresponding crude enzymes. Under denaturing-PAGE, a single proteineous band with a molecular mass of 64 kDa appeared for A. flos-aquae and S. platensis PAL. The biochemical properties of the purified PAL from both algal isolates were determined comparatively. The optimum temperature of S. platensis and A. flos-aquae PAL for forward or reverse activity was reported at 30°C, while the optimum pH for PAL enzyme isolated from A. flos-aquae was 8.9 for forward and reverse activities, and S. platensis PAL had maximum activities at pH 8.9 and 8 for forward and reverse reactions, respectively. Luckily, the purified PALs have the affinity to hydroaminate the myristicin to MMDA successfully in one step. Furthermore, a successful method for synthesis of MMDA from myristicin in two steps was also established. Gas chromatography-mass spectrometry (GC-MS) analysis was conducted to track the product formation.

Objective: The basic sources of information on drug use are epidemiological surveys, although they have some limitations: their results may be conditioned by the lack of veracity of the responses and the sampling method makes it difficult to detect lowprevalence behaviours in target populations. This study aimed to establish the epidemiological pattern of drug use in the population undergoing drug testing in hair, in the framework of judicial investigations, in order to provide an additional approach to the knowledge of high-risk drug use., Methods: A cross-sectional study on drug use was conducted on the population subjected to drug testing in hair (N=5,292) in the forensic context. Prevalence of cannabis, cocaine, heroin, ketamine, amphetamine (AP), methamphetamine (MA), 3,4-methylenedioxy- methamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxy-N-ethylamphetamine (MDEA) and methadone uses were obtained. Association between drug use and demographics, and trends of prevalence over the period were analysed using the Pearson Chi-square test. Frequency distribution of drug concentrations in hair was obtained and it was assessed in relation to gender and age using the non-parametric Mann-Whitney U and Kruskal-Wallis H methods., Results: During the period 2013-2015, prevalence of cocaine use was particularly high (49%), rating second among the population studied, after cannabis use (54%). Proportions of heroin, methadone, MDMA and amphetamine use ranged from 10% to 18%. There was a significant increase in prevalence of MDMA, heroin and amphetamine use during the period 2013-2015, as well as a significant decrease in methadone use. The rates of cannabis, cocaine and MDMA use were higher in men, whereas methadone use was higher among women., Conclusions: Cannabis and cocaine are the most frequently abused drugs among the population undergoing drug testing in hair in the framework of judicial investigations over the three-year period, although the proportions of heroin, MDMA and amphetamine users show an increasing trend. Drug use patterns vary according to age and sex, with a decrease in cannabis and MDMA use and an increase in heroin and methadone use as age increased; cannabis, cocaine and MDMA use are more prevalent among men and methadone use among women., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

Serotonin, one of the first neurotransmitters to be identified, is an evolutionarily old molecule that is highly conserved across the animal kingdom, and widely used throughout the brain. Despite this, ascribing a specific set of functions to brain serotonin and its receptors has been difficult and controversial. The 2A subtype of serotonin receptors (5-HT2A receptor) is the major excitatory serotonin receptor in the brain and has been linked to the effects of drugs that produce profound sensory and cognitive changes. Numerous studies have shown that this receptor is upregulated by a broad variety of stressors, and have related 5-HT2A receptor function to associative learning. This review proposes that stress, particularly stress related to danger and existential threats, increases the expression and function of 5-HT2A receptors. It is argued that this is a neurobiological adaptation to promote learning and avoidance of danger in the future. Upregulation of 5-HT2A receptors during stressful events forms associations that tune the brain to environmental cues that signal danger. It is speculated that life-threatening situations may activate this system and contribute to the symptoms associated with post-traumatic stress disorder (PTSD). 3,4-Methylenedioxymethamphetamine, which activates 5-HT2A receptors, has been successful in the treatment of PTSD and has recently achieved status as a breakthrough therapy. An argument is presented that 3,4-methylenedioxymethamphetamine may paradoxically act through these same 5-HT2A receptors to ameliorate the symptoms of PTSD. The central thematic contention is that a key role of serotonin may be to function as a stress detection and response system.

A 26-year-old cachectic man presented with an altered mental status. He was agitated, tremulous, hyperthermic and diaphoretic with largely dilated pupils. Collateral history revealed acute ingestion of 3,4-methylenedioxymethamphetamine on a background of chronic drug abuse. His condition deteriorated requiring sedation and intubation with transfer to the intensive care unit. A diagnosis of serotonin syndrome was made, based on his findings in keeping with the Hunter criteria, and he was treated with supportive management during a resultant and briefly sustained delirium. With gradual resolution of his agitated state, further questioning and blood work a concurrent, and potentially contributory, thyrotoxicosis was revealed. The patient was commenced on treatment for this with urgent outpatient follow-up with both a local otolaryngologist and endocrinologist for consideration of further treatment., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

Rationale: Synthetic cathinones continue to emerge in recreational drug markets worldwide. 1-(1,3-Benzodioxol-5-yl)-2-(methylamino)butan-1-one (butylone) and 1-(1,3-benzodioxol-5-yl)-2-(methylamino)pentan-1-one (pentylone) are derivatives of the cathinone compound, 1-(1,3-benzodioxol-5-yl)-2-(methylamino)propan-1-one (methylone), that are being detected in drug products and human casework., Objectives: The purpose of the present study was to examine the neuropharmacology of butylone and pentylone using in vitro and in vivo methods., Methods: In vitro uptake and release assays were carried out in rat brain synaptosomes and in cells expressing human dopamine transporters (DAT) and 5-HT transporters (SERT). In vivo microdialysis was performed in the nucleus accumbens of conscious rats to assess drug-induced changes in neurochemistry., Results: Butylone and pentylone were efficacious uptake blockers at DAT and SERT, though pentylone was more DAT-selective. Both drugs acted as transporter substrates that evoked release of [ 3 H]5-HT at SERT, while neither evoked release at DAT. Consistent with the release data, butylone and pentylone induced substrate-associated inward currents at SERT but not DAT. Administration of butylone or pentylone to rats (1 and 3 mg/kg, i.v.) increased extracellular monoamines and motor activity, but pentylone had weaker effects on 5-HT and stronger effects on motor stimulation., Conclusions: Our data demonstrate that increasing the α-carbon chain length of methylone creates "hybrid" transporter compounds which act as DAT blockers but SERT substrates. Nevertheless, butylone and pentylone elevate extracellular dopamine and stimulate motor activity, suggesting both drugs possess significant risk for abuse.

Vitreous humor (VH) shows excellent potential as a matrix of choice for postmortem analytical toxicology due to the ease of sampling and low metabolic activity. This study demonstrates a simple and rapid analytical method to identify and quantify 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxy-ethylamphetamine in VH. Samples were collected with a simple eye puncture procedure, followed by liquid-liquid extraction and derivatization with heptafluorobutyric anhydride and analysis via gas chromatography-mass spectrometry. The accuracy of the method ranged 97-103%, intra-assay precision was between 4.54 and 9.14% relative standard deviation (RSD) and interassay precision ranged from 6.92 to 10.59% RSD. Limits of detection and quantification ranged from 1.0 to 2.5 ng/mL and 10 ng/mL, respectively. The validated method was successfully applied to detect methylenedioxyamphetamine derivatives in VH samples collected from victims of fatal car crashes.

Gene therapy promises to treat diseases that arise from genetic abnormalities by correcting the underlying cause of the disease rather than treating the associated symptoms. Successful transfer of nucleic acids into cells requires efficient delivery vehicles that protect the cargo and can penetrate the appropriate cellular barriers before releasing their contents. Many viral vectors and synthetic polycationic vectors for nucleic acid delivery do not translate well from in vitro to in vivo applications due to their instability and toxicity. We synthesized and characterized a library of biocompatible low charge density polymers from a family of poly(amine- co-ester) (PACE) terpolymers produced via enzyme catalyzed polymerization. PACE polymers are highly customizable; we found that the terpolymer composition can be optimized to produce efficient transfection of various nucleic acids-including DNA plasmids, mRNA, and siRNA-in specific cell types with low toxicity. Our findings suggest that the unique tunability of PACEs offers new tools for gene therapy and other biomedical applications.

The number of emerging novel stimulants modified based on beta-keto variations of amphetamine-like substances continues to rise. Dibutylone reports described in the medical and toxicological literature are limited, therefore little information is available in terms of quantitative confirmation or metabolism. During this study, authentic human specimens, including blood, urine, vitreous humor, oral fluid and liver were quantitatively and qualitatively analyzed for the presence of dibutylone and butylone, with paired case history and demographic information. Dibutylone concentrations were variable across all specimen types, specifically ranging from 10 to 1,400 ng/mL in postmortem blood specimens. The metabolic profile of dibutylone was mapped by in vitro incubation with human liver microsomes (HLM). Samples were analyzed using a SCIEX TripleTOF® 5600+ quadrupole time-of-flight mass spectrometer. Data processing was conducted using MetabolitePilot™. Authentic human specimens, including blood, urine, vitreous humor, oral fluid and liver, were utilized for in vivo verification of five HLM-generated metabolites in analytically confirmed cases of dibutylone use. Butylone was confirmed as a metabolite of dibutylone, but issues involving co-ingestion of these two novel stimulants or potential co-existence from synthesis lead to ineffectiveness as a true biomarker. Hydrogenation of the beta-ketone of dibutylone resulted in the most prominent metabolite found in human specimens, and its uniqueness to dibutylone over other stimulants leads to its classification as an appropriate biomarker for dibutylone ingestion. This is the first study to map the metabolic profile of dibutylone, including verification in authentic specimens, confirming metabolic conversion to butylone and identifying biomarkers more useful in forensic toxicological drug testing.

Monitoring population drug use through wastewater-based epidemiology (WBE) is a useful method to quantitatively follow trends and estimate total drug consumption in communities. Concentrations of drug biomarkers might be low in wastewater due to dilution; and therefore analysis of pooled urine (PU) is useful to detect consumed drugs and identify targets of illicit drugs use. The aims of the study were (1) to screen PU and urinated soil (US) samples collected at festivals for illicit drug excretion products using hyphenated techniques; (2) to develop and validate a hydrophilic interaction liquid chromatography - mass spectrometry / mass spectrometry (HILIC-MS/MS) method of quantifying urinary targets of identified drugs in wastewater; and (3) to conduct a 24 h stability study, using PU and US to better reflect the chemical environment for targets in wastewater. Cocaine (COC) and ecstasy-like compounds were the most frequently detected illicit drugs; an analytical method was developed to quantify their excretion products. Hydroxymethoxymethamphetamine (HMMA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), HMMA sulfate (HMMA-S), benzoylecgonine (BE), and cocaethylene (CE) had 85-102% of initial concentration after 8 h of incubation, whereas COC and ecgonine methyl ester (EME) had 74 and 67% after 8 h, respectively. HMMA showed a net increase during 24 h of incubation (107% ± 27, n = 8), possibly due to the cleavage of HMMA conjugates, and biotransformation of MDMA. The results suggest HMMA as analytical target for MDMA consumption in WBE, due to its stability in wastewater and its excretion as the main phase I metabolite of MDMA. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

New psychoactive "designer drugs" are synthetic compounds developed to provide similar effects to illicit drugs of abuse, but not subjected to legal control. The rapidly changing legal status of novel psychoactive drugs triggers the development of new compounds, analogs of well-known amphetamine or mescaline. New designer drugs used as substitutes in ecstasy pills are the least investigated and can cause life-threatening effects on users. The aim of our research was to examine the effects of acute administration of 4-methoxyamphetamine (PMA, 5 and 10 mg/kg), 4-methoxy-N-methylamphetamine (PMMA, 5 and 10 mg/kg), and mephedrone (MEPH, 5, 10 and 20 mg/kg) on extracellular and tissue level of dopamine (DA), 5-hydroxytryptamine (5-HT) and their metabolites in rat brain, by microdialysis method in freely moving animals and HPLC. Similarly to 3,4-methylenedioxymethamphetamine (MDMA, 5 and 10 mg/kg) PMA, PMMA and MEPH enhanced the release of DA and 5-HT in rat striatum, nucleus accumbens, and frontal cortex. DA tissue content was increased by MEPH and PMMA in striatum, by MEPH, PMA, and PMMA in nucleus accumbens, and by PMA in frontal cortex. Instead, cortical DA level was decreased by MEPH and PMMA. MEPH did not influence 5-HT tissue level in striatum and nucleus accumbens, but decreased its level in frontal cortex. PMMA increased 5-HT content in striatum, while PMA enhanced it in nucleus accumbens and frontal cortex. Observed changes in brain monoamines and their metabolites by new psychoactive drugs suggest that these drugs may be capable of development of dependence. Further experiments are needed to fully investigate the neurotoxic and abuse potential of those drugs.

Objective: To explore the relationship of gentamicin-induced cochlear damage with autophagy-related protein LC3, beclin1, Na(+-)K(+-)2Cl(-) cotransporter (NKCC1) mRNA and endothelin-1 (ET-1), and investigate the protective mechanism of PPTA against gentamicin-induced cochlear damage., Methods: Sixty guinea pigs were randomly divided into control group (with saline and artificial perilymph injections), model group (with gentamicin and artificial perilymph injections), concurrent treatment group (with gentamicin and PPTA injections), model control group (with artificial perilymph injection 7 days after gentamicin injection) and delayed treatment group (with PPTA injection 7 days after gentamicin injection). Saline and gentamicin (160 mg/kg) were injected intraperitoneally, and artificial perilymph and PPTA were injected into the otocysts on a daily basis for 7 consecutive days. Hearing impairment of the guinea pigs was analyzed with ABR, and the protein expressions of beclin1 and LC3 in cochlear tissue were tested. The expression of NKCC1 mRNA was detected with RT-PCR, and the expression of ET-1 was detected immunohistochemically., Results: The ABR thresholds in the model group and model control group were similar (P>0.05) , but significantly higher than those in the other 3 groups (P<0.05); the threshold was significantly lower in concurrent treatment group than in delayed treatment group (P<0.05). Compared with those in the other 4 groups, the expressions of LC3 II, beclin1, and NKCC1 mRNA were significantly increased in the model group (P<0.05); and those in delayed treatment group were significantly lower than those in the model control group (P<0.05). The expressions of ET-1 in the Corti organ, striavascularis and spiral ganglion were significantly higher in the model group but significantly lower in the control group than those in the other 4 groups; ET-1 expression was significantly lower in delayed treatment group than in the model control group., Conclusion: PPTA offers protection against gantamicin-induced cochlear damage in guinea pigs by inhibiting cell autophagy and suppressing of NKCC1 and ET-1 expressions. Early intervention with PPTA produces better therapeutic effect, suggesting that gantamicin causes irreversible injury of the auditory cells.

In recent years, the abuse of synthetic cathinones has increased considerably. This study proposes a method, based on gas chromatography/mass spectrometry (GC-MS), to analyze and quantify six synthetic cathinones in urine samples: mephedrone (4-MMC), methylone (bk-MDMA), butylone, ethylone, pentylone and methylenedioxypyrovalerone (MDPV). In our procedure, the urine samples undergo solid-phase extraction (SPE) and derivatization prior to injection into the GC-MS device. Separation is performed using a HP-5MS capillary column. The use of selective ion monitoring (SIM mode) makes it is good sensitivity in this method, and the entire analysis process is within 18 min. In addition, the proposed method maintains linearity in the calibration curve from 50 to 2,000 ng/mL (r(2) > 0.995). The limit of detection of this method is 5 ng/mL, with the exception of MDPV (20 ng/mL); the limit of quantification is 20 ng/mL, with the exception of MDPV (50 ng/mL). In testing, the extraction performance of SPE was between 82.34 and 104.46%. Precision and accuracy results were satisfactory <15%. The proposed method was applied to six real urine samples, one of which was found to contain 4-MMC and bk-MDMA. Our results demonstrate the efficacy of the proposed method in the identification of synthetic cathinones in urine, with regard to the limits of detection and quantification. This method is highly repeatable and accurate., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Phenethylamine-based designer drugs are prevalent within the new psychoactive substance market. Characterisation of their metabolites is important in order to identify suitable biomarkers which can be used for better monitoring their consumption. Careful design of in vitro metabolism experiments using subcellular liver fractions will assist in obtaining reliable outcomes for such purposes. The objective of this study was to stepwise investigate the in vitro human metabolism of seven phenethylamine-based designer drugs using individual families of enzymes. This included para-methoxyamphetamine, para-methoxymethamphetamine, 4-methylthioamphetamine, N-methyl-benzodioxolylbutanamine, benzodioxolylbutanamine, 5-(2-aminopropyl) benzofuran and 6-(2-aminopropyl) benzofuran. Identification and structural elucidation of the metabolites was performed using liquid chromatography-quadrupole-time-of-flight mass spectrometry. The targeted drugs were mainly metabolised by cytochrome P450 enzymes via O-dealkylation as the major pathway, followed by N-dealkylation, oxidation of unsubstituted C atoms and deamination (to a small extent). These drugs were largely free from Phase II metabolism. Only a limited number of metabolites were found which was consistent with the existing literature for other phenethylamine-based drugs. Also, the metabolism of most of the targeted drugs progressed at slow rate. The reproducibility of the identified metabolites was assessed through examining formation patterns using different incubation times, substrate and enzyme concentrations. Completion of the work has led to a set of metabolites which are representative for specific detection of these drugs in intoxicated individuals and also for meaningful evaluation of their use in communities by wastewater-based drug epidemiology., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Ebola virus disease (EVD) is one of the most lethal transmissible infections characterized by a high fatality rate, and a treatment has not been developed yet. Recently, it has been shown that cationic amphiphiles, among them the antiarrhythmic drug amiodarone, inhibit filovirus infection. In the present work, we investigated how amiodarone interferes with Ebola virus infection. Wild-type Sudan ebolavirus and recombinant vesicular stomatitis virus, pseudotyped with the Zaire ebolavirus glycoprotein, were used to gain further insight into the ability of amiodarone to affect Ebola virus infection. We show that amiodarone decreases Ebola virus infection at concentrations close to those found in the sera of patients treated for arrhythmias. The drug acts by interfering with the fusion of the viral envelope with the endosomal membrane. We also show that MDEA, the main amiodarone metabolite, contributes to the antiviral activity. Finally, studies with amiodarone analogues indicate that the antiviral activity is correlated with drug ability to accumulate into and interfere with the endocytic pathway. Considering that it is well tolerated, especially in the acute setting, amiodarone appears to deserve consideration for clinical use in EVD., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Autophagic (type II) cell death, characterized by the massive accumulation of autophagic vacuoles in the cytoplasm of cells, has been suggested to play pathogenetic roles in cerebral ischemia, brain trauma, and neurodegenerative disorders. 3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is an illicit drug causing long-term neurotoxicity in the brain. Apoptotic (type I) and necrotic (type III) cell death have been implicated in MDMA-induced neurotoxicity, while the role of autophagy in MDMA-elicited neurotoxicity has not been investigated. The present study aimed to evaluate the occurrence and contribution of autophagy to neurotoxicity in cultured rat cortical neurons challenged with MDMA. Autophagy activation was monitored by expression of microtubule-associated protein 1 light chain 3 (LC3; an autophagic marker) using immunofluorescence and western blot analysis. Here, we demonstrate that MDMA exposure induced monodansylcadaverine (MDC)- and LC3B-densely stained autophagosome formation and increased conversion of LC3B-I to LC3B-II, coinciding with the neurodegenerative phase of MDMA challenge. Autophagy inhibitor 3-methyladenine (3-MA) pretreatment significantly attenuated MDMA-induced autophagosome accumulation, LC3B-II expression, and ameliorated MDMA-triggered neurite damage and neuronal death. In contrast, enhanced autophagy flux by rapamycin or impaired autophagosome clearance by bafilomycin A1 led to more autophagosome accumulation in neurons and aggravated neurite degeneration, indicating that excessive autophagosome accumulation contributes to MDMA-induced neurotoxicity. Furthermore, MDMA induced phosphorylation of AMP-activated protein kinase (AMPK) and its downstream unc-51-like kinase 1 (ULK1), suggesting the AMPK/ULK1 signaling pathway might be involved in MDMA-induced autophagy activation.

The α4β2 nicotinic acetylcholine receptor (nAChR) is a molecular target of 3,4-methylenedioxymethamphetamine (MDMA), a synthetic drug also known as ecstasy, and it modulates the MDMA-mediated reinforcing properties. However, the enantioselective preference of the α4β2 nAChR subtype still remains unknown. Since the two enantiomers exhibit different pharmacological profiles and stereoselective metabolism, the aim of this study is to assess a possible difference in the interaction of the MDMA enantiomers with this nAChR subtype. To this end, we report a novel simple, yet highly efficient enantioselective synthesis of the MDMA enantiomers, in which the key step is the diastereoselective reduction of imides derived from optically pure tert-butylsulfinamide. The enantioselective binding to the receptor is examined using [(3)H]epibatidine in a radioligand assay. Even though the two enantiomers induced a concentration-dependent binding displacement, (S)-MDMA has an inhibition constant 13-fold higher than (R)-MDMA, which shows a Hill's coefficient not significantly different from unity, implying a competitive interaction. Furthermore, when NGF-differentiated PC12 cells were pretreated with the compounds, a significant increase in binding of [(3)H]epibatidine was found for (R)-MDMA, indicating up-regulation of heteromeric nAChR in the cell surface. Finally, docking and molecular dynamics studies have been used to identify the binding mode of the two enantiomers, which provides a structural basis to justify the differences in affinity from the differential interactions played by the substituents at the stereogenic centre of MDMA. The results provide a basis to explore the distinct psychostimulant profiles of the MDMA enantiomers mediated by the α4β2 nAChR subtype., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Objective: To study the protective effect of peperphentonamine hydrochloride (PPTA) against gentamicin-induced cochlear damage and its mechanism to inhibit cell apoptosis., Methods: Guinea pigs with normal hearing were randomized into control, gentamicin, and PPTA treatment groups, and the guinea pigs models of gentamicin-induced cochlear damage received intraperitoneal injection of PPTA. The changes of hearing of the guinea pigs were evaluated with auditory brainstem response (ABR) test, and the protein expression of caspase-3 in the cochlear tissue was detected using Western blotting. TUNEL staining, scanning and transmission electron microscopy were performed to observe the morphological changes of the cochlea., Results: The threshold in ABR in PPTA treatment group was significantly higher than that in the control group (P<0.05) but significantly lower than that in gentamicin group. Western blotting showed a significantly increased caspase-3 expression in gentamicin group (P<0.001); caspase-3 expression in PPTA group was obviously higher than that in the control group but much lower than that in gentamicin group (P<0.001). TUNEL assay and electron microscopy revealed serious damages of the hair cells in gentamicin group with numerous apoptotic cells in the organ of Corti, stria vascularis and spiral ganglion, and such cochlear damages were obviously alleviated in PPTA group., Conclusion: PPTA can protect against gentamicin-induced cochlear damage in guinea pigs by decreasing the protein expression of caspase-3 to inhibit cell apoptosis.

MDMA (3,4-methylenedioxymethamphetamine) is a substituted amphetamine and popular drug of abuse. Its mood-enhancing short-term effects may prompt its consumption under stress. Clinical studies indicate that MDMA treatment may mitigate the symptoms of stress disorders such as posttraumatic stress syndrome (PTSD). On the other hand, repeated administration of MDMA results in persistent deficits in markers of serotonergic (5-HT) nerve terminals that have been viewed as indicative of 5-HT neurotoxicity. Exposure to chronic stress has been shown to augment MDMA-induced 5-HT neurotoxicity. Here, we examine the transcriptional responses in the hippocampus to MDMA treatment of control rats and rats exposed to chronic stress. MDMA altered the expression of genes that regulate unfolded protein binding, protein folding, calmodulin-dependent protein kinase activity, and neuropeptide signaling. In stressed rats, the gene expression profile in response to MDMA was altered to affect sensory processing and responses to tissue damage in nerve sheaths. Subsequent treatment with MDMA also markedly altered the genetic responses to stress such that the stress-induced downregulation of genes related to the circadian rhythm was reversed. The data support the view that MDMA-induced transcriptional responses accompany the persistent effects of this drug on neuronal structure/function. In addition, MDMA treatment alters the stress-induced transcriptional signature.

Objective: To investigate the relationship between IL-1β and TNF-α mRNA and Fas protein expressions and cochlear ischemia reperfusion injury and investigate the protective mechanism of PPTA against cochlear reperfusion injury., Methods: Sixty-four guinea pigs were randomly divided into normal control group, blank control group, ischemia/reperfusion (by clamping the bilateral vertebral artery and right common carotid artery for 1 h) control group, and ischemia/reperfusion with PPTA treatment group. In PPTA group, PPTA was injected via the femoral vein immediately after reperfusion, and ischemia/reperfusion control group received saline injection. In 6 guinea pigs from each group, the cochlear tissues were removed after 24 h of reperfusion for examination of expressions of IL-1β and TNF-α mRNA by real-time PCR, and the rest animals were used for immunohistochemical detection of Fas protein., Results: Compared with those of normal group and blank control group, the expressions of IL-1β and TNF-β mRNA increased significantly after cochlear ischemia/reperfusion (P<0.001), but were lowered significantly by PPTA (P<0.001). Positive expression of Fas protein expression was detected in the Corti organ, spiral ganglion and stria vascularis in ischemia/reperfusion control group with significantly higher IOD values than those of the other 3 groups (P<0.05). The IOD value showed no significant difference between PPTA-treated group, normal control group, and blank control group (P>0.05)., Conclusions: PPTA can suppress the expression of Fas protein and IL-1β and TNF-β mRNAs in the cochlea of guinea pigs with cochlear ischemia/reperfusion. The protective effect of PPTA against cochlear ischemia/reperfusion is mediated probably by inhibition of inflammatory responses and cell apoptosis.

In 2010, an internet snapshot of EMCDDA anticipated the presence of 5-iodo-2-aminoindan (5-IAI) within the recreational drug market. In 2011, this compound, a psychoactive derivative of 2-aminoindane, was identified in recreational products sold in the United Kingdom. 5-IAI is a rigid analogue of p-iodoamphetamine producing MDMA-like effects. The aim of this paper is to summarize the clinical, pharmacological, and toxicological information about this new potential drug of abuse., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The calcium sensitizers levosimendan and piperphentonamine hydrochloride (PPTA) are used as cardiovascular drugs for treatment of heart failure. Given that levosimendan has been reported to exhibit a neuroprotective profile in a model of traumatic brain injury, it was interesting to know whether PPTA, a new calcium sensitizer recently developed in China, exerts a similar effect. The objective of this study was to determine whether PPTA exhibited neuroprotective effects and whether these properties were associated with memory. Four-vessel occlusion (4-VO) was used to induce global cerebral ischemia/reperfusion injury in rats treated with or without PPTA (5, 10 mg/kg, i.p., 2 h after the onset of reperfusion and then once a day for 15 consecutive days). Memory was measured using the step-through passive avoidance test. Neurochemical changes were examined in rat PC12 cells treated with oxygen-glucose deprivation (OGD) for 4 h followed by reoxygenation (OGD-R) for 24 h, in the absence or presence of PPTA. In vehicle-treated animals, 4-VO for 10 min produced memory deficits, as demonstrated by decreased retention in step-through passive avoidance, and massive neuron loss in the hippocampal CA1 subregion. These effects were attenuated by PPTA. The results were consistent with those observed in PC12 cells. PPTA treatment increased cell viability, as indicated by MTT assay, inhibited apoptosis, and decreased extracellular lactate dehydrogenase levels in Na(2)S(2)O(4)-treated PC12 cells. These results provide novel demonstration for the ability of PPTA to attenuate cerebral ischemia-induced memory deficits via neuroprotection in the hippocampus. The neuroprotective effect of PPTA appears to be associated with its anti-apoptotic activity. PPTA has the therapeutic potential for ischemic stroke.

Piperphentonamine hydrochloride (PPTA) is a new calcium sensitizer. A liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for determination of piperphentonamine and its metabolites M1 and M6 was developed for the first time and applied to a pharmacokinetics study. Protein precipitation was used for pre-treatment of plasma samples, and solid phase extraction method was used for pre-treatment of urine samples. The chromatographic separation was achieved on a C(18) column using gradient elution in this study: A: 1% acetic acid aqueous solution, and B: acetonitrile. The whole analysis lasted for 10.5min and the gradient flow rate was 0.25mL/min constantly. The detection was performed of a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via a positive electrospray ionization source. The results were that the m/z ratios of monitored precursor ions and product ions of PPTA, M1 and M6 were 354.0→191.8, 356.0→148.7 and 358.0→148.7, respectively. From the standard curve, the concentration ranges of both PPTA and M1 in blood and urine samples were 0.1-500ng/mL and 0.1-200ng/mL, respectively; the concentration ranges of M6 in blood sample and urine sample were 0.2-500ng/mL and 0.2-200ng/mL, respectively; and the correlation coefficient of standard curve was r>0.99. A total of 31 healthy Chinese subjects participated in the pharmacokinetic study of single bolus intravenous injection of piperphentonamine hydrochloride. They were divided into three dosage groups and given 0.2, 0.4 and 0.6mg/kg of PPTA. After drug administration, concentrations of PPTA, M1 and M6 in human plasma and urine samples were determined to evaluation the pharmacokinetic characteristics of PPTA and its metabolites M1 and M6., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The easily available "legal highs", which are products containing psychoactive substances, such as cathinones, piperazines and synthetic cannabinoids, are abused by adolescents in Poland and in the world as alternatives to classic drugs, such as amphetamines or marijuana. The majority of these potentially dangerous substances are still legal and they are associated with a risk of severe poisoning or even death, and provide new challenges in clinical and forensic toxicological practice. Investigations in the field of "designer drugs" may be well illustrated by the case of a suicide of a 21-year old male who ingested a specified dose of a preparation called "Amphi-bi-a" that contains bk-MBDB, chemically 2-methylamino-1-(3,4-methylenedioxyphenyl) butan-1-one, which belongs to the cathinone group, as a synthetic euphoric empathogen and psychoactive stimulant that is chemically similar to MDMA. It is one of more common components of "legal highs" examined in Poland and other countries. The documentation of the case includes a clinical assessment of the patient's health status performed during his almost 4-h hospitalization before death, autopsy and histological examinations supported by toxicological findings revealing bk-MBDB at extremely high concentrations (at 20 mg/l in the blood and 33 mg/kg in the liver); hence, this body of evidence contributes to knowledge in the field of "designer drugs". Inventions of designers of new psychoactive xenobiotics, which are much in demand, especially in view of the dynamic Internet marketing, which drums up narcobusiness, must be balanced by a national strategy developed by medical, legal and educational circles in the modern civilized world in order to prevent the spreading of the phenomenon., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background and Purpose: Here, we have compared the neurochemical profile of three new cathinones, butylone, mephedrone and methylone, in terms of their potential to inhibit plasmalemmal and vesicular monoamine transporters. Their interaction with 5-HT and dopamine receptors and their psychostimulant effect was also studied., Experimental Approach: Locomotor activity was recorded in mice following different doses of cathinones. Monoamine uptake assays were performed in purified rat synaptosomes. Radioligand-binding assays were carried out to assess the affinity of these compounds for monoamine transporters or receptors., Key Results: Butylone, mephedrone and methylone (5-25 mg·kg(-1) ) caused hyperlocomotion, which was prevented with ketanserin or haloperidol. Methylone was the most potent compound inhibiting both [(3) H]5-HT and [(3) H]dopamine uptake with IC(50) values that correlate with its affinity for dopamine and 5-HT transporter. Mephedrone was found to be the cathinone derivative with highest affinity for vesicular monoamine transporter-2 causing the inhibition of dopamine uptake. The affinity of cathinones for 5-HT(2A) receptors was similar to that of MDMA., Conclusions and Implications: Butylone and methylone induced hyperlocomotion through activating 5-HT(2A) receptors and increasing extra-cellular dopamine. They inhibited 5-HT and dopamine uptake by competing with substrate. Methylone was the most potent 5-HT and dopamine uptake inhibitor and its effect partly persisted after withdrawal. Mephedrone-induced hyperlocomotion was dependent on endogenous 5-HT. Vesicular content played a key role in the effect of mephedrone, especially for 5-HT uptake inhibition. The potency of mephedrone in inhibiting noradrenaline uptake suggests a sympathetic effect of this cathinone., (© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.)

Different elimination was reported for the two enantiomers of the designer drug 3,4-methylenedioxyethylamphetamine (MDEA) in vivo. In the present work, the enantioselectivity of glucuronidation and sulfation of the MDEA phase I metabolites 3,4-dihydroxyethylamphetamine (DHEA) and 4-hydroxy-3-methoxyethylamphetamine (HMEA) was investigated. First, glucuronide standards were synthesized using rat liver microsomes. Incubations were performed with recombinant human UDP-glucuronyltransferases (UGT) and pooled human liver microsomes (pHLM) for glucuronidation and using recombinant human sulfotransferases (SULT) and pooled human liver cytosol (pHLC) for sulfation. Product formation experiments were performed by quantification of the phase II metabolites using liquid chromatography-high-resolution mass spectrometry. Additionally, substrate depletion experiments were conducted by gas chromatography-mass spectrometry after chiral derivatization for sulfation. UGT2B7, 2B15, and 2B17 were involved in glucuronidation of HMEA and SULT1A1 and SULT1A3 and SULT1A3 and SULT1E1 in the sulfation of DHEA and HMEA, respectively. SULTs provided much higher affinity, whereas UGTs showed higher capacities. Marked stereoselectivity could be observed for UGT2B15, UGT2B17, and pHLM toward S-HMEA, for SULT1A3 and pHLC toward S-DHEA and for SULT1A3 and pHLC toward R-HMEA. In conclusion, the phase II metabolism might also contribute to the observed different pharmacokinetic properties of MDEA., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Previous studies have demonstrated high removal rates of amphetamine-type-stimulants (ATSs) through conventional drinking water treatments; however the behaviour of these compounds through disinfection steps and their transformation into disinfection-by-products (DBPs) is still unknown. In this work, for the first time, the reactivity of some ATSs such as amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyethylamphetamine (MDEA) with chlorine has been investigated under simulated and real drinking water treatment conditions in order to evaluate their ability to give rise to transformation products. Two new DBPs from these illicit drugs have been found. A common chlorinated-by-product (3-chlorobenzo)-1,3-dioxole, was identified for both MDA and MDEA while for MDMA, 3-chlorocatechol was found. The presence of these DBPs in water samples collected through drinking water treatment was studied in order to evaluate their formation under real conditions. Both compounds were generated through treatment from raw river water samples containing ATSs at concentration levels ranging from 1 to 15 ng/L for MDA and from 2.3 to 78 ng/L for MDMA. One of them, (3-chlorobenzo)-1,3-dioxole, found after the first chlorination step, was eliminated after ozone and GAC treatment while the MDMA DBP mainly generated after the postchlorination step, showed to be recalcitrant and it was found in final treated waters at concentrations ranging from 0.5 to 5.8 ng/L., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Introduction: A new generation of designer phenethylamines have emerged and aggressively marketed as "legal highs." The drugs are labeled "not for human consumption" to avoid widespread recognition and prosecution under the existing analog drug laws. The newest generation includes methylone and butylone. Methylone and butylone have minor structural changes and similar pharmacodynamics properties to scheduled drugs., Case Report: We report a case of a healthy 24-year-old who ingested a capsule containing methylone and butylone sold as "Ecstasy" at a concert. The patient presented to the emergency department, comatose febrile, tachycardic, tachypnic, and hypertensive. On exam, she was diaphoretic, tremulous, hyperreflexic, and had sustained clonus. The patient was aggressively cooled, and despite maximal supportive care, the patient progressed to multi-system organ failure and ultimately expired. We obtained and analyzed both her urine and a capsule found on her person similar to the capsules ingested. In both samples, laboratory analysis identified only methylone and butylone., Conclusion: This is the first reported death for methylone or butylone and the first human or animal ingestion of butylone. Clinicians and public health officials should work together as new designer drugs emerge.

A novel method is described for the extraction of methamphetamine, amphetamine, and methylenedioxyphenylalkylamine designer drugs, such as 3,4-methylenedioxy-methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxyethylamphetamine, N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine, and 3,4-(methylenedioxyphenyl)-2-butanamine, from human whole blood using molecularly imprinted solid-phase extraction as highly selective sample clean-up technique. Whole blood samples were diluted with 10 mmol/L ammonium acetate (pH 8.6) and applied to a SupelMIP-Amphetamine molecularly imprinted solid-phase extraction cartridge. The cartridge was then washed to eliminate interferences, and the amphetamines of interest were eluted with formic acid/methanol (1:100, v/v). After derivatization with trifluoroacetic anhydride, the analytes were quantified using gas chromatography-mass spectrometry. Recoveries of the seven amphetamines spiked into whole blood were 89.1-102%. The limits of quantification for each compound in 200 μL of whole blood were between 0.25 and 1.0 ng. The maximum intra- and inter-day coefficients of variation were 9.96 and 13.8%, respectively. The results show that methamphetamine, amphetamine, and methylenedioxyphenylalkyl-amine designer drugs can be efficiently extracted from crude biological samples such as whole blood by molecularly imprinted solid-phase extraction with good reproducibility. This extraction method will be useful for the pretreatment of human samples before gas chromatography-mass spectrometry., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: To investigate the effect of piperphentonamine hydrochloride (PPTA) on cognitive deficits induced by ischemia-reperfusion and explore the possible mechanisms., Methods: SD rats were randomly divided into sham-operated group, ischemia-reperfusion group (with saline injection), PPTA-treated groups (2.5, 5, 10 mg/kg) and edaravone-treated group (6 mg/kg). Cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion, and the agents were administrated 1 h after ischemia. At 24 h after ischemia, step-through passive avoidance test was carried out, and 24 h later IL-1β, TNF-α, caspase-3 and HSP-70 mRNA expressions in the ischemic brain tissues were measured with RT-PCR., Results: In the step-through passive avoidance test, the rats in the ischemia-reperfusion group showed significantly shorter latency and more error times than those in the sham group, and these behavioral changes were improved significantly by treatments with PPTA and edaravone. Cerebral ischemia-reperfusion caused significantly increased expressions of IL-1β, TNF-α, caspase-3 and HSP-70 mRNA, and these changes were obviously reversed by PPTA, but not by edaravone., Conclusions: PPTA can reverse cognitive deficits induced by cerebral ischemia-reperfusion probably by decreasing the inflammatory responses and cell apoptosis in the brain, suggesting its potential as a new therapeutic agent for improving the cognitive function following cerebral ischemia-reperfusion.

3,4-Methylenedioxymethamphetamine (MDMA)'s O-demethylenated metabolite, 3,4-dihydroxymethamphetamine (HHMA), has been hypothesized to serve as a precursor for the formation of toxic catechol-thioether metabolites (e.g., 5-N-acetylcystein-S-yl-HHMA) that mediate MDMA neurotoxicity. To further test this hypothesis, HHMA formation was blocked with dextromethorphan (DXM), which competitively inhibits cytochrome P450 enzyme-mediated O-demethylenation of MDMA to HHMA. In particular, rats were randomly assigned to one of four treatment groups (n = 9-12 per group): (1) Saline/MDMA; (2) DXM/MDMA; (3) DXM/Saline; (4) Saline/Saline. During drug exposure, time-concentration profiles of MDMA and its metabolites were determined, along with body temperature. One week later, brain serotonin (5-HT) neuronal markers were measured in the same animals. DXM did not significantly alter core temperature in MDMA-treated animals. A large (greater than 70%) decrease in HHMA formation had no effect on the magnitude of MDMA neurotoxicity. These results cast doubt on the role of HHMA-derived catechol-thioether metabolites in the mechanism of MDMA neurotoxicity., (Copyright © 2011 Wiley-Liss, Inc.)

N-hydroxy-3,4-methylenedioxymethamphetamine (N-OH-MDMA) is a psychedelic illicit drug that has recently been circulating in Japan. The aims of this study were (i) to optimise enzymatic hydrolysis conditions of the conjugated forms of N-OH-MDMA and its demethylated metabolite N-hydroxy-3,4-methylenedioxyamphetamine (N-OH-MDA), (ii) to investigate the urinary excretion profiles of N-OH-MDMA in rats, and (iii) to compare urinary excretion profiles of N-OH-MDMA and 3,4-methylenedioxymethamphetamine (MDMA). Conjugated forms of the N-hydroxylated compounds (N-OH-MDMA and N-OH-MDA) were almost successfully hydrolysed to their nonconjugated forms under anaerobic conditions after helium purging of the solution. The sum of N-OH-MDMA and N-OH-MDA was used to evaluate the amount of excreted N-hydroxylated metabolites because of degradation of N-OH-MDMA to N-OH-MDA during hydrolysis. Up to 24 h after oral administration of N-OH-MDMA oxalate, the main urinary metabolites were MDMA (14.3% of dose) and 3,4-MDA (7.7% of dose). Most of the N-hydroxylated forms were excreted as glucuronide conjugates. The total amount of N-hydroxylated metabolites after hydrolysis was 1.1% of dose. Urinary excretion profiles of MDMA were similar to that of N-OH-MDMA. It may be difficult to differentiate between abuse of MDMA and N-OH-MDMA by urine analysis.

To facilitate the analysis of targeted drugs under high sample volume testing environment, an extraction, derivatization and gas chromatographic-mass spectrometric analysis method was developed for simultaneously determination of amphetamine (AMP), methamphetamine (MAMP), 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyethylamphetamine (MDEA), ketamine, and norketamine in urine. This method utilized solid-phase extraction in conjunction with derivatization using heptafluorobutyric anhydride (HFBA) as the derivatization reagent. Using a 1-mL sample, the limits of quantitation achieved for the analysis of AMP, MAMP, MDA, MDMA, MDEA, ketamine, and norketamine were 25, 15, 60, 60, 70, 25, and 30 ng/mL, respectively. Upper limits of quantitation were 8000 ng/mL for all amphetamines and 6000 ng/mL for ketamine and norketamine. Except for dehydronorketamine (DHNK), within-day and between-day precisions (as expressed in CV%) for quality control samples were ≤ 3.1% and ≤ 4.95%, respectively. Except DHNK, the within-day accuracy ranged between 96.0% and 110.7% and the between-day accuracy ranged between 96.9% and 108.7%. A group of 107 urine samples previously determined to contain the target analytes were analyzed by this new approach. Quantitative data produced by both methods agreed well. With this new approach, we were able to use a single analytical protocol to conduct the confirmation test for samples that preliminarily tested positive (by immunoassay) for amphetamines, ketamine, or both.

The recreational use of the so-called "legal-highs" has been in both the medical and political arena over the last year as a result of the appearance of "head shops" in many towns in Ireland. These shops specialized in selling new psychotropic compounds that circumvented established drug legislation. Little is known about the potentially harmful effects of these substances but case reports suggest a plethora of harmful psychological and physical effects. Our case describes for the first time acute liver failure associated with the ingestion of two of these amphetamine type compounds.

Aims: Our group has conducted several Internet investigations into the biobehavioural effects of self-reported recreational use of MDMA (3,4-methylenedioxymethamphetamine or Ecstasy) and other psychosocial drugs. Here we report a new study examining the relationship between self-reported Ecstasy use and traces of MDMA found in hair samples., Methods: In a laboratory setting, 49 undergraduate volunteers performed an Internet-based assessment which included mood scales and the University of East London Drug Use Questionnaire, which asks for history and current drug use. They also provided a hair sample for determination of exposure to MDMA over the previous month., Results: Self-report of Ecstasy use and presence in hair samples were consistent (p < 0.00001). Both subjective and objective measures predicted lower self-reported ratings of happiness and higher self-reported stress. Self-reported Ecstasy use, but not presence in hair, was also associated with decreased tension., Conclusion: Different psychoactive drugs can influence long-term mood and cognition in complex and dynamically interactive ways. Here we have shown a good correspondence between self-report and objective assessment of exposure to MDMA. These data suggest that the Internet has potentially high utility as a useful medium to complement traditional laboratory studies into the sequelae of recreational drug use., (Copyright © 2010 S. Karger AG, Basel.)

This study evaluated the potential for partial separation of drugs from their deuterated internal standards using Cerex(®) Polycrom™ CLIN II solid-phase extraction (SPE) cartridges. After elution from the column and derivatization, gas chromatography-mass spectrometry results showed that the target compound eluted from the SPE cartridge prior to its deuterated form. This elution separation effect was greater for 3,4-methylenedioxymethamphetamine (MDMA) and methamphetamine (MAMP) than for the other drugs studied. When the drugs were eluted in 0.5 mL increments from a 50 mg sorbent bed, no drug appeared in the first fraction. The drug to internal standard ratios (expected value 1.00) for subsequent fractions collected were 1.30, 1.07, and 0.83 for MDA/MDA-d(5); 1.65, 1.18, 0.67, and 0.56 for MDMA/MDMAd(5); and 1.37, 1.18, and 0.95 for MDEA/MDEA-d(6). For d-AMP and d-MAMP, the expected ratio was 0.40. The subsequent ratios were 0.63, 0.46, 0.35, and 0.34 for d-AMP/d-AMP-d(11); and 1.00, 0.59, 0.25, and 0.18 for d-MAMP/d-MAMP-d(14). The affinity of d-MAMPd(14) was shown to be greater than that of d-MAMP-d(5), and deuteration at the propyl end of the molecule was shown to increase binding more than deuteration on the phenyl group.

Three regioisomeric 3,4-methylenedioxyphenethylamines having the same molecular weight and major mass spectral fragments of equal mass have been reported as drugs of abuse in forensic studies in recent years. These compounds are 3,4-methylenedioxy-N-ethylamphetamine (MDEA), 3,4-methylenedioxy-N,N-dimethylamphetamine (MDMMA), and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB). A series of seven additional side chain regioisomers have mass spectra essentially equivalent to the three controlled drug substances, all have molecular weight of 207 and major fragment ions in their electron ionization mass spectra at m/z 72 and 135/136. The trifluoroacetyl, pentafluoropropionyl, and heptafluorobutryl derivatives of the primary and secondary regioisomeric amines were evaluated in GC-MS studies. The mass spectra for these derivatives were significantly individualized, and the resulting unique fragment ions allowed for specific side chain identification. The trifluoroacetyl and heptafluorobutryl derivatives provided more specific fragment ions for molecular individualization among these regioisomeric substances. These perfluoroacyl derivatives showed reasonable resolution on the polar stationary phase Rtx-200.

On November 25, 2008, the U.S. Department of Health and Human Services posted a final notice in the Federal Register authorizing the use of liquid chromatography-tandem mass spectrometry (LC-MS-MS) and other technologies in federally regulated workplace drug testing (WPDT) programs. To support this change, it is essential to explicitly demonstrate that LC-MS-MS, as a technology, can produce results at least as valid as gas chromatography (GC)-MS, the long-accepted standard in confirmatory analytical technologies for drugs of abuse. A series of manufactured control urine samples (n = 10 for each analyte) containing amphetamine, methamphetamine, (±)-3,4-methylenedioxyamphetamine, (±)-3,4-methylenedioxymethamphetamine, (±)-3,4-methylenedioxyethylamphetamine, phencyclidine, and (±)-11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol at concentrations ranging from 10% to 2000% of federal cutoffs were analyzed with replication by five federally regulated laboratories using GC-MS and at RTI International using LC-MS-MS. Interference samples as described in the National Laboratory Certification Program 2009 Manual were analyzed by GC-MS and LC-MS-MS as well as previously confirmed urine specimens of WPDT origin. Matrix effects were assessed for LC-MS-MS. Results indicated that LC-MS-MS analysis produced results at least as precise, accurate, and specific as GC-MS for the analytes investigated in this study. Matrix effects, while evident, could be controlled by the use of matrix-matched controls and calibrators with deuterated internal standards.

In recent years, a new class of designer drugs has appeared on the drugs of abuse market in many countries, namely, the so-called beta-keto (bk) designer drugs such as mephedrone (bk-4-methylmethamphetamine), butylone (bk-MBDB), and methylone (bk-MDMA). The aim of the present study was to identify the metabolites of mephedrone in rat and human urine using GC-MS techniques and to include mephedrone, butylone, and methylone within the authors' systematic toxicological analysis (STA) procedure. Six phase I metabolites of mephedrone were detected in rat urine and seven in human urine suggesting the following metabolic steps: N-demethylation to the primary amine, reduction of the keto moiety to the respective alcohol, and oxidation of the tolyl moiety to the corresponding alcohols and carboxylic acid. The STA procedure allowed the detection of mephedrone, butylone, methylone, and their metabolites in urine of rats treated with doses corresponding to those reported for abuse of amphetamines. Besides macro-based data evaluation, an automated evaluation using the automated mass spectral deconvolution and identification system was performed. Mephedrone and butylone could be detected also in human urine samples submitted for drug testing. Assuming similar kinetics in humans, the described STA procedure should be suitable for proof of an intake of the bk-designer drugs in human urine.

An N-hydroxy analogue of 3,4-methylendioxymethamphetamine (MDMA), N-hydroxy MDMA (N-OH MDMA), has recently been distributed as a new designer drug in some drug markets. Very little data is available to the metabolic and pharmacological properties of N-OH MDMA, although it has been reported that the N-demethyl analogue, N-hydroxy-3,4-methylenedioxyamphetamine (N-OH MDA), is mainly metabolized to MDA in rats. In this study, an analytical method for the determination of N-OH MDMA and its metabolites in biological samples was developed, and the metabolic properties of N-OH MDMA in rats were investigated. After the i.p. administration of N-OH MDMA to pigmented hairy rats (5mg/kg/day, 10 days), N-OH MDMA and its N-dehydroxy and N-demethyl metabolites (MDMA, N-OH MDA and MDA) in rat plasma, urine and hair samples were determined by ultra-performance LC (UPLC)-MS/MS. The hair sample was extracted by 1-h sonication and overnight soaking in 5M hydrochloric acid-methanol (1:20). The plasma, urine, and hair extract samples were purified using a solid-phase extraction procedure. N-OH MDMA in the samples could be precisely analyzed by avoiding an alkaline environment. The parent compound very rapidly disappeared from the rat plasma (<15min) and urine (<10h), and most of the N-OH MDMA was excreted in the rat urine as MDMA and MDA in 72h. In the rat hair samples collected 4 weeks after the first administration, N-OH MDMA (0.03ng/mg) and N-OH MDA (0.13ng/mg) were clearly detected as well as MDMA (149ng/mg) and MDA (52ng/mg). This analytical method will be useful for the analysis of N-OH MDMA and its metabolites in biological samples.

HPLC and TLC methods were developed for separation and detection of some amphetamine analogs: methamphetamine (MA); 3,4-methylenedioxymethamphetamine (MDMA, "ecstasy"); and 3,4-methylenedioxy-N-ethylamphetamine (MDEA) in spiked plasma samples. The methods are based on purple chromogens formed by displacement reaction of these secondary aliphatic amine-bearing drugs with 7,7,8,8-tetracyanoquinodimethane at 80 degrees C for 25 min. For HPLC, both normal phase (silica gel) and RP (C18) columns were used. With the former, good detection limits in plasma were obtained with a 6 min run: 70, 100, and 500 ng/mL for MDMA, MA, and MDEA, respectively. For TLC, hexane-chloroform (1 + 9) and benzene-diethyl ether-petroleum ether (40-60 degrees)-acetonitrile-ethyl methyl ketone (2 + 3.5 + 3.5 + 0.5 + 0.5) were used as mobile phases for silica gel 60 TLC and cyano-bonded silica gel HPTLC plates, respectively. The former offered more sensitive results than the latter. Influence of evaporation steps on recovery and interferences for the HPLC and TLC methods were investigated. The developed methods are selective, simple, and easily applicable.

Active-site water molecules form an important component in biological systems, facilitating promiscuous binding or an increase in specificity and affinity. Taking water molecules into account in computational approaches to drug design or site-of-metabolism predictions is currently far from straightforward. In this study, the effects of including water molecules in molecular docking simulations of the important metabolic enzyme cytochrome P450 2D6 are investigated. The structure and dynamics of water molecules that are present in the active site simultaneously with a selected substrate are described, and based on this description, water molecules are selected to be included in docking experiments into multiple protein conformations. Apart from the parent substrate, 11 similar and 53 dissimilar substrates are included to investigate the transferability of active-site hydration sites between substrates. The role of water molecules appears to be highly dependent on the protein conformation and the substrate.